Plants have ancient classes of reproductive and vegetative actin genes with distinct expression patterns. During the last grant period we have examined the hypothesis that these ancient actin genes were preserved throughout vascular plant evolution because they have distinct patterns of regulation and/or because they express functionally distinct actin protein isovariants. We have extended our research to include the profiling and actin-related protein (ARP) families, and have identified more than 200 actin binding proteins (ABP) encoded by the Arabidopsis genome. Mutations in actin, profiling, and ARP genes have resulted in a variety of informative phenotypes ranging from those affecting cell, organ, and plant size to extreme morphological differences including alterations in cell polarity, sterility, and embryo lethality. We have explored the importance of actin protein isovariants by making numerous subclass- and isovariant-specific monoclonal antibodies to examine their distinct expression patterns. We have demonstrated that ectopic expression, but not overexpression, of actin isovariants dramatically disrupts the development of most organs and tissues During the next grant period we will apply a wide variety of cell biological, molecular, and genetic approaches to our research dissecting the roles of different isovariants of the actins, ARPs, and ABPs. Our new Specific Aims are: 1) to characterize the phenotypes of Arabidopsis actin, ARP, and profiling mutants and combinations of mutants focusing on the most distinct functions of these genes; 2) to investigate the functional significance of various actin isovariants to organ, tissue, and cell development; 3) to identify the significance of organ and tissue specific expression of actin-binding protein isovariants (ABPs) (e.g., profiling, ADF), and 4) to construct plants with a minimal complement of reproductive and vegetative isovariants to further characterize differences among actin and ABP isovariants.